CMB lab exam guide - Summary Introductory Medical Microbiology PDF

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Lab exam 1 study guide...

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CMB Lab Exam Study Guide 1 ● Topics on exam

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Review Lab protocol ○ No food or drink in lab ○ Closed toed shoes ○ Wash hands and wipe down bench before and after ○ Be prepared, read instructions beforehand ○ Label samples ○ Take good notes

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Aseptic Technique- approach used in class to avoid contamination of cultures and people ○ Wash hands and wipe down benches before and after class ○ Flame loop, needles, and mouth of tubes prior and after obtaining samples of bacteria ○ Flame top of tube before streaking slant in tube ○ Dip spreader in alcohol and flame before using to spread ○ Have flame close to plates to reduce time ○ Never put plate cover, cap, or cotton plug on bench ○ Limit time exposed to air for any culture ○ Wear gloves

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Isolation of Bacteria ○ Be able to diagram and explain step by step how to streak for isolation ■ Using aseptic technique ■ Divide plate into sections ■ Apply bacteria from sample in first section ■ Flame loop ■ Drag bacteria from first section to second and spread around plate ■ Flame loop ■ Drag bacteria from second section to third section ■ Close plate ○ Why we streak for isolation ■ Separate and isolate bacteria so we know we have a pure culture

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Stains used in lab ○ Gram Stain ■ Differential stain used to distinguish two or more types of microorganisms based on physical or chemical interactions with the chemicals used ■ Takes advantage of differences in bacterial cell wall characteristics ● Differentiates between gram + and gram ■ Label and diagram a gram + and gram - bacterial cell wall ■ Explain steps of gram stain 1. Crystal violet= primary stain a. Stains peptidoglycan wall 2. Lugol’s iodine= mordant a. Complexes the crystal violet with peptidoglycan 3. alcohol= decolorizing agent a. Disrupts outer membrane and washes away the crystal violet of a GRAM -, leaving it colorless b. Dehydrates the thick PG layer effectively trapping crystal violet in a GRAM +, leaving it purple 4. Safranin= secondary stain or counter stain a. Stain the GRAM - cells pink

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Stains used in the lab ○ Simple stain ■ Used to visualize cells and endospores ■ Crystal violet ○ Shaeffer-Fulton method ■ Visualize endospores ● Malachite green= primary stain ● heating= mordant to allow the primary stain to penetrate ● Decolorize = water ● safranin= counterstain

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Use and Care of Microscope ○ Carry by holding base and arm ○ Only use oil on 100x ○ NEVER pass through 40x with oil on ○ Focus condenser and oculars ○ Dab 100x objective with lens paper until oil is gone before storing in cabinet

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Cellular morphology of bacteria using microscope ○ Bacilli or rods are long thin shaped cells ■ Bacilli in a long chain are streptobacilli ■ Bacilli in a parallel or picket fence are palisades ○ Cocci- spherical cells ■ Cocci in pairs are diplococci ■ Cocci in chains are streptococci ■ Cocci in clusters are staphylococci ○ A flagellum-locomotive appendage of bacteria ■ Not all bacteria have flagella ■ Very few cocci are motile ■ Monotrichous: single flagellum ■ Lophotrichous: having 2+ flagella at one or both ends ■ Amphitrichous: single flagellum on opposite ends ■ Peritrichous: flagella project in all directions ○ Bacterial endospores ■ Dormant and resistant structures produced by some bacteria ■ Made mostly by Gram + rods ■ Help to ensure the survival of a bacterium through periods of stress (starvation, radiation, heat, desiccation)

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Test for different enzyme use to breakdown substances for their utilization ○ Alpha-amylase breaks down starch and starch products ■ Lugol’s iodine reacts with starch to produce a dark color and leaves a colorless halo if starch is broken down into dextrins ○ Gelatinase cleaves gelatin into amino acids ■ Hydrochloric acid is used to develop the frazier’s gelatin plates because unhydrolyzed gelatin precipitates with the addition of acid, causing a clear halo ○ Caseinase breaks down the milky protein casein ■ The intact casein gives the media a milky appearance ■ Presence of caseinase leads to a clear halo around caseinase positive colonies

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Detecting the products of fermentation ○ Acids ■ Detected by use of pH indicators in media (such as Brom Cresol Purple, which turns yellow in acid) ○ Alcohols ■ Difficult to detect ○ Gases ■ Detected with the use of a Durham tube, which is a small tube inverted in a larger tube of broth. Gases produced by bacteria form gas bubbles in the tube

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Other enzymes used by bacteria ○ Catalase is an enzyme that converts hydrogen peroxide into water and oxygen gas ■ Hydrogen peroxide- a toxic metabolite of oxygen metabolism ○ Oxidase ■ Use oxygen to make water and powers the synthesis of ATP (energy units of the cell) ○ Trophtophanase ■ Indole side chain of tryptophan can be cleaved off by some types of bacteria (via), we can detect this process by testing for the presence of indole in a tryptophan media ○ Cysteine desulfurase ■ The sulfur side chain of cysteine removed enzymatically to produce hydrogen sulfide ■ Detect for process by presence of hydrogen sulfide in a peptone-iron tube

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Other Reactions ○ Dissimilatory nitrate reduction ■ Induced by anaerobic conditions ■ Ability of some bacteria to use nitrogen as a final electron acceptor instead of oxygen ■ This process proceeds through some combination of the following steps: nitrate, nitrite, nitrogen gas, or ammonia ○ MR-VP citrate test is used to distinguish enterobacter and E.coli ■ MR= methyl red ● pH indicator that is red in acidic conditions ● This indicates E. Coli is present and making acid ■ VP= Voges-Proskauer method ● Allow for detection of butanediol pathway ○ VP test tests for acetyl methyl carbinol= acetoin ■ Which is a product of the butanediol pathway ■ Enterobacter can use citrate as a sole carbon source, while E. Coli does not ● If citrate is used: heavy growth and/or the bromothymol blue in the media will go from green to blue...